The invention relates to novel transferrin binding proteins of Pasteurella haemolytica, truncations, analogs, homologs and isoforms thereof; nucleic acid molecules encoding the proteins and truncations, analogs, and homologs of the proteins; vaccines containing the proteins; antibodies against the proteins; and, uses of the proteins and nucleic acid molecules.
Members of the genus Pasteurella comprise a group of related bacterial species that are important pathogens of ruminants. This group includes the species Pasteurella haemolytica which has been classified into two biotypes, A and T, on the basis of sugar utilization, and into 16 serotypes which are recognized on the basis of their somatic antigens (Biberstein, E. L. et al., 1960; Fraser et al., 1982). The T-type strains of P. haemolytica, characterized by utilization of trehalose, have been recently reclassified as a new species P. trehalosi (Sneath, P. H. A. et al., 1990).
Pneumonic pasteurellosis caused by Pasteurella haemolytica is a major economic problem to the cattle, sheep and goat industries world-wide. Shipping fever, a variation of this disease, is a major problem in the cattle industry in North America and is almost exclusively caused by type A1 strains of this species (Babiuk, L. A. and S. D. Acres, 1984). Serotype A2 is the most prevalent disease-causing type in sheep but other serotypes may be important in sheep and goats (Gilmour and Gilmour, 1991). The related species, Pasteurella trehalosi (formerly know as T-type P. haemolytica) is the causitive agent of septicemia in lambs, a problem plaguing the sheep industry particularly in the United Kingdom. Similarly, strains of the related species Pasteurella multocida, are responsible for haemorrhagic septicemia, a serious infection in cattle and water buffalo, which is particularly serious in South East Asia.
Vaccination is a desired method of control for pasteurellosis in ruminants but success has been limited by the lack of immunizing preparations that induce protection against all disease-causing serotypes, particularly if a vaccine effective for all ruminants is considered. Killed whole cell vaccines elicited inconsistent levels of protection and antibody response in calves (Wilkie, B. N., 1980). Homologous vaccines containing sodium salicylate extracts (SSEs) protected sheep against diseases due to serotypes A1, A6 and A9 (Gilmour et al., 1983) but not against the more epidemic serotype A2 (Fraser et al., 1982). An exotoxin produced by P. haemolytica which is specifically lethal to leucocytes and alveolar macrophages from ruminants (Benson et al., 1978) has shown a lot of promise as a vaccine candidate in protection experiments in calves and sheep (13,35) but there is limited protection against heterologous serotypes (33). The inclusion of proteins induced under iron-limited growth conditions into a vaccine for pasteurellosis in lambs has been implicated in enhanced protection (15).
Previous studies have established that the ability of pathogenic bacteria to acquire iron in vivo is a critical factor in their pathobiology (7,11). One mechanism of iron retrieval from the host iron-binding glycoprotein, transferrin, involves direct binding of transferrin by surface receptors on the bacteria and the removal of iron from transferrin and uptake into the cell (21). Schryvers (1992) describes the isolation of transferrin receptor proteins from various bacterial pathogens using affinity chromatography. The transferrin receptor has been shown to consist of two proteins, called transferrin binding protein 1 or A (Tbp1 or TbpA) and transferrin binding protein 2 or B (Tbp2 or TbpB). The receptor-mediated type of iron uptake has been demonstrated to operate in serotype A bovine strains of P. haemolytica (26). Cells of P. haemolytica growing in vitro under iron-limited conditions express a number of iron-repressible outer membrane proteins (IROMPs) identical to those produced by cells recovered in vivo from infected sites in animals with pasteurellosis (9,10). Especially prominent among these proteins were those of molecular sizes 100, 77, 70 and 60 Kda (9,10). The 100 Kda protein has been identified as one of the host specific transferrin receptors in bovine isolates (26) while some of the other IROMPs had been suggested as possibly associated with the 100 Kda protein in an iron acquisition receptor complex (26). The role of the IROMPs expressed by P. haemolytica from lambs (10) in iron acquisition has not been elucidated, neither is it known if similar proteins are expressed by goat isolates.
P. haemolytica acquires iron from bovine host transferrin by a receptor-mediated type of mechanism. The proposal that bacteria with this type of iron acquisition mechanism may be solely dependent upon their surface receptor for iron acquisition in vivo (29) implies that they can only cause disease in those hosts whose transferrin is recognized by their surface receptors. P. haemolytica has been reported to cause disease in cattle, sheep and goats and accordingly their surface receptors would be expected to recognize these hosts"" transferrins. Therefore it is important to determine whether sheep and goat isolates also possessed transferrin receptors involved in iron acquisition, to evaluate their specificities for different ruminant transferrins and to determine if there is antigenic relatedness amongst the surface receptors from the different strains causing pneumonic pasteurellosis in cattle, sheep and goats.
Transferrin receptors were identified in a collection of Pasteurella haemolytica (and P. trehalosi) strains of various serotypes and biotypes (A and T) from cattle, sheep and goats. Growth studies, binding studies and affinity isolation experiments demonstrated that these receptors had identical specificities which recognized transferrins from cattle, sheep and goats. This indicates that there are conserved regions on the receptor proteins, involved in ligand binding, which are accessible at the cell surface.
Antisera prepared against the individual purified receptor proteins (TbpA and TbpB) from a serotype A1 strain of P. haemolytica demonstrated considerable crossreactivity against receptor proteins from a representative selection of strains. The cross-reactivity was also observed against intact cells indicating that there are conserved immunological epitopes at the cell surface which could serve as targets for the host""s immune effector mechanisms.
The present inventors have cloned, sequenced and expressed tbpA and tbpB genes encoding the transferrin receptor proteins, TbpA and TbpB (also referred to herein as Tbp1 and Tbp2, respectively), from Pasteurella haemolytica A1. The genes were organized in an operon arrangement of tbpB- tbpA. The tbpB gene was preceded by putative promoter and regulatory sequences, and followed by a 96 base pair intergenic sequence in which no promoter regions were found, suggesting that the two genes are coordinately transcribed. The deduced amino acid sequences of the TbpA and TbpB proteins had regions of homology with the corresponding Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae and Actinobacillus pleuropneumoniae Lbp and Tbp proteins. The intact tbpB gene was expressed in a T7 expression system and the resulting recombinant TbpB protein retained the functional bovine transferrin binding characteristics. The availability of the recombinant TbpB enabled the inventors to demonstrate its specificity for ruminant transferrin, its ability to bind both the C-and N-terminal lobes of bovine transferrin, and its preference for the iron-loaded form of this protein.
The present inventors also significantly found that vaccination with a formulation containing P. haemolytica TbpA and TbpB provided significant protection against experimental bovine pneumonic pasteurellosis. Immunization with two doses of TbpB also provides protection
Broadly stated, the present invention provides a purified and isolated nucleic acid molecule comprising a sequence encoding a TbpA protein, or a purified and isolated nucleic acid molecule comprising a sequence encoding a TbpB protein. The TbpA and TbpB proteins bind ruminant transferrins and function in receptor-mediated iron acquisition by P. haemolytica in its ruminant hosts. The TbpA protein is approximately 100 kDa, and TbpB is approximately 60 kDa in size.
In an embodiment of the invention, the purified and isolated nucleic acid molecules comprise a sequence encoding a TbpA protein having the amino acid sequence as shown in FIG. 22 or SEQ.ID.NO:2, or a sequence encoding a TbpB protein having the amino acid sequence as shown in FIG. 24 or SEQ.ID.NO:4. In a preferred embodiment of the invention, the purified and isolated nucleic acid molecules comprise a sequence encoding a TbpA protein and having the nucleic acid sequence as shown in FIG. 21 or SEQ.ID.NO:1, or a sequence encoding a TbpB protein having the nucleic acid sequence as shown in FIG. 23 or SEQ.ID.NO:3.
The invention also contemplates (a) nucleic acid molecules comprising a sequence encoding a truncation of TbpA or TbpB which is unique to the protein, an analog or homolog of TbpA or TbpB or a truncation thereof, (herein collectively referred to as xe2x80x9cTbpA related proteinsxe2x80x9d or xe2x80x9cTbpB related proteinsxe2x80x9d, respectively); (b) a nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the full length nucleic acid encoding TbpA or TbpB having the amino acid sequences as shown in FIGS. 22 and 24 respectively, or to a TbpA or TbpB related protein; (c) a nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the full length nucleic acid sequence of the tbpA or tbpB genes having the sequences as shown in FIGS. 21 or SEQ.ID.NO:1, or FIG. 23 or SEQ.ID.NO:3, respectively.
The invention further contemplates a purified and isolated double stranded nucleic acid molecule containing a nucleic acid molecule of the invention, hydrogen bonded to a complementary nucleic acid base sequence.
The nucleic acid molecules of the invention may be inserted into an appropriate expression vector, i.e. a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence. Accordingly, recombinant expression vectors adapted for transformation of a host cell may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively linked to the nucleic acid molecule.
The recombinant expression vector can be used to prepare transformed host cells expressing TbpA and/or TbpB, or a TbpA or a TbpB related protein. Therefore, the invention further provides host cells containing a recombinant molecule of the invention.
The invention further provides a method for preparing a novel TbpA or TbpB, and TbpA or TbpB related proteins, utilizing the purified and isolated nucleic acid molecules of the invention. In an embodiment a method for preparing TbpA or TbpB is provided comprising (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of TbpA or TbpB; and (d) isolating the recombinant TbpA or TbpB.
The invention further broadly contemplates a purified and isolated TbpA or TbpB which binds to ruminant transferrin, preferably obtained by culturing a host cell containing a recombinant expression vector of the invention. In an embodiment of the invention, a purified TbpA or TbpB is provided which has the amino acid sequence as shown in FIG. 22 or FIG. 24 respectively. The invention also includes truncations of the protein and analogs, homologs, and isoforms of the protein and truncations thereof (i.e., xe2x80x9cTbpA or TbpB related proteinsxe2x80x9d).
The TbpA and TbpB, or TbpA and TbpB related proteins of the invention may be conjugated with other molecules, such as proteins, to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
The invention further contemplates antibodies having specificity against an epitope of TbpA or TbpB, or TbpA or TbpB related proteins of the invention. Antibodies may be labelled with a detectable substance and they may be used to detect the TbpA or TbpB, or TbpA or TbpB related proteins of the invention in samples.
The invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and accordingly to TbpA or TbpB, or TbpA or TbpB related proteins of the invention. Thus, the invention also relates to a probe comprising a sequence encoding TbpA or TbpB, or TbpA or TbpB related proteins. The probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nudeotide sequence coding for a protein which displays one or more of the properties of TbpA or TbpB.
The invention still further provides a method for identifying a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, comprising reacting TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, with at least one substance which potentially can bind with TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related proteins, or an activated form thereof, and assaying for complexes, for free substance, for non-complexed TbpA or TbpB or a TbpA or TbpB related proteins, or an activated form thereof. Substances which potentially can bind TbpA or TbpB, or TbpA or TbpB related proteins, include transferrins, particularly ruminant transferring, analogs and derivatives of transferrins and antibodies against TbpA and TbpB, or TbpA or TbpB related proteins.
Still further, the invention provides a method for assaying a medium for the presence of an agonist or antagonist of the interaction of TbpA or TbpB, or TbpA or TbpB related proteins, and a substance which binds to TbpA or TbpB, or TbpA or TbpB related proteins or an activated form thereof. In an embodiment, the method comprises providing a known concentration of TbpA or TbpB, or TbpA or TbpB related proteins, with a substance which is capable of binding to TbpA or TbpB, or TbpA or TbpB related proteins and a suspected agonist or antagonist substance under conditions which permit the formation of complexes between the substance and TbpA or TbpB, or TbpA or TbpB related proteins, and assaying for complexes, for free substance, for non-complexed TbpA or TbpB, or TbpA or TbpB related proteins. In a preferred embodiment of the invention, the substance is a ruminant transferrin, analog, derivative or part thereof or an antibody against TbpA or TbpB, or TbpA or TbpB related proteins.
Substances which affect expression of TbpA or TbpB, or TbpA or TbpB related proteins, may also be identified using the methods of the invention by comparing the pattern and level of expression of TbpA or TbpB, or TbpA or TbpB related proteins of the invention, in cells in the presence, and in the absence of the substance.
The substances identified using the methods of the invention may be used in the treatment of animals, particularly ruminants infected with P. haemolytica and accordingly they may be formulated into pharmaceutical compositions for adminstration to ruminants, such as cattle, sheep and goats suffering from infection with P. haemolytica or exposed to infection by P. haemolytica. 
The present inventors have demonstrated that the TbpA or TbpB, or TbpA or TbpB related proteins of the invention, are immunogenic. Therefore, the invention also relates to antibodies against the TbpA or TbpB, or TbpA or TbpB related proteins of the invention. In an embodiment, the antibodies are cross reactive against TbpA or TbpB or TbpA, or TbpB related proteins, from a wide range of serotypes of P. haemolytica. The antibodies may be used in the diagnosis and treatment of P. haemloytica infection and may be used, for example, in passive immunization to treat or prevent diseases in ruminants caused by P. haemolytica. 
The invention further includes vaccine compositions comprising the TbpA or TbpB, or TbpA or TbpB related proteins of the invention, either alone, or in combination. The invention still further includes methods of immunizing a host, preferably a ruminant host against infection by P. haemolytica by administering therapeutically effective amounts of such vaccines. The present inventors have demonstrated that different strains of P. haemolytica, from a range of ruminants, are able to bind and utilize a range of ruminant transferrins. Thus it is contemplated that the vaccine compositions of the invention will be useful as broad spectrum vaccines suitable for immunizing a range of ruminants, such as sheep, cows and goats against infection with a wide range of P. haemolytica biotypes and serotypes.
The invention also contemplates the use of nucleic acid molecules of the invention encoding TbpA or TbpB, or TbpA or TbpB related proteins, in a recombinant viral vector vaccine for augmenting the immune response of a ruminant to P. haemolytica or for treating P. haemolytica infection. Recombinant viral vectors may be constructed using techniques known in the art.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.